Notwithstanding intensive research for a vaccine in the 14 years since the discovery and characterization of HIV, major obstacles remain for HIV vaccine and immunotherapy development. These hurdles include HIV-1 variability, a lack of understanding of the virus structure, and a lack of understanding of the immune responses necessary for prevention of HIV infection. See D. Burton and J. Moore, Nature Medicine, 1998, 4:495-48. The head of the US government's AIDS vaccine research committee stated on Feb. 1, 1998 that a safe vaccine to prevent AIDS could still be more than a decade away from testing, because too much remains unknown about how the body's immune system works (http://cnn.cm/HEALTH/9802/01/aids.vaccine.search).
There was early optimism for efficacious recombinant HIV-1 envelope subunit vaccines (e.g., gp120 and gp160 vaccine products) given that vaccinee sera from several clinical trials were capable of neutralizing laboratory isolates of HIV-1 in vitro (Belshe et al., J. Am. Med. Assoc., 1994, 272:475; Keefer et al., AIDS Res. Hum Retroviruses, 1994, 10:1713). This optimism was shaken when the vaccinee sera were found to be largely ineffective in neutralizing HIV-1 primary patient isolates (Hanson, AIDS Res. Hum Retroviruses, 1994, 10:645; Mascola et al., J Infect Dis., 1996, 173:340). These disappointing findings led NIH to decide in June 1994 to postpone costly large-scale efficacy trials of several recombinant envelope protein based HIV subunit vaccines.
HIV vaccine research now focuses on primary isolates which are believed to more closely resemble HIV strains responsible for human infection than do the commonly used laboratory strains (Sawyer et al., J Virol, 1994, 68:1342; Wrin et al., J Virol, 1995, 69:39). Primary isolates of HIV-1 are obtained by limited cultivation of patient PBMCs or plasma with uninfected PBMCs. Primary viruses can be readily distinguished by phenotype as discussed below from the T cell line adapted (TCLA) viruses such as IIb/LAI, SF2, and MN, which have been passaged over time in human T-lymphoid cell lines and have become well-adapted to grow in these T cell lines:
(1) Unlike TCLA viruses, most primary isolates do not readily grow in T cell lines.
(2) Unlike TCLA viruses which are all syncytium- inducing, primary isolates include both syncytium-inducing (SI) isolates that induce syncytium formation in PBMC culture and non-syncytium-inducing (NSI) isolates. Among the SI primary isolates, most will replicate in the especially HIV-sensitive T cell line MT2, but few can replicate in the less permissive T cell lines such as CEM or H9 that are commonly used for the culture of TCLA isolates. Non-syncytium-inducing (NSI) primary isolates replicate only in primary T cells.
(3) Primary isolates are highly resistant to in vitro neutralization by recombinant soluble forms of the viral receptor protein CD4 (rsCD4) requiring 200-2700 times more rsCD4 than TCLA strains for comparable neutralization (Daar et al., PNAS USA, 1990, 87:6574-6578).
(4) Primary isolates are also resistant to neutralizing antibodies elicited by the use of gp120 (envelope) vaccines. In contrast, The TCLA strains are sensitive to neutralization by antibodies with specificities for the viral envelope (Sawyer et al., J Virol, 1994, 68:1342; and, Mascola et al., 1996).
These phenotypic characteristics of primary isolates are due to poorly understood structural features of HIV, particularly the inaccessible quality of the viral envelope with respect to anti-env antibodies (D. Burton and J. Moore, Nature Medicine, 1998, 4:495-498). Viral variability, a genotypic characteristic, also remains as an obstacle to the development of HIV vaccines of worldwide efficacy (Mascola et al., 1996). These factors together account for the unexpected failure of virally-directed AIDS vaccines which were developed against readily grown TCLA homotypic strains. An alternative approach to HIV vaccine development could be by intervention on the HIV receptors of the host cell, thereby blocking infection by preventing HIV from binding to or fusing with susceptible cells. The cell-directed approach offers methods to overcome the hypervariability of the HIV envelope and phenotypic diversity.
A cell-directed approach for protection from HIV infection was suggested by active and passive immunization studies in the SIV rhesus macaque model which showed that anti-cell antibodies greatly contributed to protection from infection (Stott, Nature, 1991, 353:393). In addition, monoclonal antibodies directed against CD4, a T cell receptor for MHC Class II molecules and the primary receptor for HIV binding, have long been known to block infection in HIV-1 neutralization assays in a manner that is dependent on the CD4 epitope, not the virus strain (Sattentau et al., Science, 1986; 234:1120). Of particular relevance for a cell-directed approach to immunoprophylaxis, anti-CD4 monoclonal antibodies have been effective in blocking infection of cells by primary isolates (Daar et al., Proc. Natl. Acad. Sci. USA, 1990; 87: 6574; and Hasunuma et al., J Immunol., 1992; 148:1841). Other potentially effective cell-directed approaches can be to target chemokine receptors CXCR4, CCR5, CCR2b, and CCR3 that recently have been identified as coreceptors for HIV (Feng et al., Science, 1996; 272:872; and, Doranz et al., Cell, 1996; 85:1149). These coreceptors function together with CD4 to initiate post-binding interactions of the viral envelope glycoprotein with the host cell membrane and in post-entry steps of retrovirus replication (Chackerian et al., J Virol, 1997; 71:3932). The requirement for both CD4 and a coreceptor for efficient HIV binding and fusion suggests that either or both of these molecules may be good targets for cell-directed strategies to inhibit infection. Antibodies directed to a host cell CD4/coreceptor complex have been shown to affect both binding and post-binding steps of HIV infection (Wang, WO 97/46697). These antibodies neutralized virus-to-cell or cell-to-cell transmission of both syncytium-inducing (SI) and non-SI (NSI) strains of HIV. A chemokine antagonist that binds to CCR5 has also been shown to be effective in preventing infection by both SI and NSI viruses (Simmons et al., Science, 1997; 276:276). Neutralization of NSI isolates is particularly significant as NSI strains are believed to be responsible for most HIV transmission and are frequently resistant to anti-HIV antibodies which neutralize TCLA isolates (Fauci, 1996). The agents that target the cellular receptors of HIV avoid the need to confront diverse phenotypes and the hypervariability of the viral envelope, and in addition offer potential neutralization activity against HIV-2 and SIV (Chen et al, J Virol, 1997; 71:2705; Pleskoff et al., J Virol, 1997; 71:3259; WO 97/46697).
A host cell receptor/coreceptor complex comprising CD4 and a chemokine coreceptor on the surface of the host T cells, which facilitates viral binding and entry into the host T cells, is reported to be an effective target for neutralizing antibodies in a co-pending patent application (U.S. Ser. No. 08/657,149 also published as WO 97/46697). In that application, the present inventor demonstrated that except for antibodies directed against this cell surface antigen complex, no other anti-cell antibodies raised in response to cell surface antigens on HPB-ALL cells neutralized HIV-1 primary isolates. Antibodies with the desired properties as described in that application can block infection of monkeys by SIV, in vivo HIV-1 infection of the human immune system reconstituted in mice, in vitro infections of human cells by HIV-1 primary isolates of diverse phenotypes and genotypes and block infection of human cells by HIV-2. This cell surface antigen complex comprising the CD4 receptor associated with a chemokine coreceptor (CD4/coreceptor complex) acts as a target for protective anti-cell antibodies.
Anti-cell antibodies to the CD4/coreceptor complex display a more effective pattern of neutralization against relevant HIV strains than do anti-virus antibodies directed against the viral envelope. As shown in the co-pending application (WO 97/46697), a monoclonal antibody (MAb B4), produced against HPB-ALL and having a moderate reactivity against the recombinant soluble CD4 (rsCD4) protein and a strong binding to the HPB-ALL cells, i.e., with specificity for the CD4/coreceptor cell surface complex, was found highly effective in neutralizing primary isolates of HIV-1 but less effective in neutralizing TCLA strains. In contrast, anti-env antibodies display the reverse pattern for preferential neutralization of TCLA strains.
It was found that MAb B4 neutralized HIV primary isolates in an in vitro microplaque assay at a concentration of &lt;10 .mu.g/ml. In contrast, polyclonal antibodies with high titer (&gt;5 Log.sub.10) specificity for recombinant soluble CD4 (rsCD4) failed to display any neutralizing activity for HIV primary isolates despite their strong T cell binding activities. Thus, the primary isolates appear to be preferentially sensitive to the antibody with specificity for the cell surface CD4/coreceptor antigen complex, in comparison to antibodies with a pure CD4 specificity. The extensive characterization of HIV neutralization by anti-CD4/coreceptor complex antibodies includes MAb B4 and its homologs MAb M2 and MAb B13 (WO 97/46697).
The mechanism for the broad neutralizing activity of antibodies to the CD4/coreceptor complex is unclear because of the diverse roles of that cell surface complex in mediating HIV infection, as shown by the ability of those antibodies to affect both binding and post-binding steps of HIV infection (Wang, WO 97/46697). The CD4/coreceptor cell surface complex may play dual roles in mediating HIV infection and pathogenesis: (1) as a T cell surface receptor for HIV binding, cell fusion and entry by HIV; or (2) as an HIV suppressive factor.
However effective as agents for the inhibition of HIV infection, the above cell-directed antagonists or antibodies, including highly neutralizing antibodies with specificity for the host cell CD4/coreceptor complex, cannot be used as preventative vaccines. They are agents for passive immunization. For efficacy, these agents must be frequently administered so as to maintain serum concentrations sufficient for full receptor occupancy. A vaccine that acts by inducing an active anti-self antibody response against the CD4/coreceptor complex, by active immunization, would be preferable for protective immunity. Such a vaccine, if it can be developed, would provide effective and long term protection from infection by the infrequent and convenient administration of small quantities of immunogen.
For efficacy, the immunogenic components of such a vaccine must mimic relevant sites on the host cell receptor/coreceptor complex with fidelity sufficient to evoke cross-inhibitory antibodies, while retaining site-specificity sufficient to avoid adverse immunosuppression. The identification of such sites for mimicry by synthetic antigens has not been disclosed by the available anti-cell antibodies which neutralize HIV, including anti-CD4 antibodies with neutralizing activity. For example, a anti-CD4 monoclonal antibody reported to be neutralizing (Burkly et al., J Immunol, 1992; 149:1779) and the broadly neutralizing anti-CD4/coreceptor monoclonal antibody reported by Wang (WO 97/46697) recognize discontinuous conformational sites on CD4 that cannot be readily duplicated. Also, the reproduction of useful host cell antigenic target sites as portions of long recombinant immunogens cannot be readily applied as a means to avoid the need for exact knowledge of the vulnerable sites. Most antibodies raised by immunization with CD4 lack useful specificities (Davis et al., Nature, 1992; 358:76). For example, high titer hyperimmune antiserum to rsCD4 was devoid of neutralizing activity for primary isolates of HIV (WO 97/46697). Moreover, antibodies with broad reactivity for extensive regions of a T cell antigen are expected to be overly immunosuppressive, in contrast to a site-specific antibody (Reimann et al., AIDS Res. Hum Retroviruses, 1997; 13: 933). In addition, although extensive mapping studies of CD4 have yielded a structure function map for the molecule (Sattentau et al., Science, 1986, 234:1120; Peterson and Seed, Cell, 1988, 54:65; Jameson et al., Science, 1988, 240:1335; Sattentau et al., J Exp. Med., 1989, 170:1319; Hasunuma et al., J Immunol, 1992, 148:1841; Burkly et al., J Immunol, 1992, 149:1779; Davis et al., Nature, 1992, 358:76), this mapping does not provide for structural models of sufficient precision for prediction of vulnerable effector sites that are duplicable as synthetic peptides. The available models for CD4 do not disclose useful CD4-based immunogens.
Furthermore, however effective as agents for the inhibition of HIV infection, the cell-directed antagonists or antibodies previously discussed, such as the highly neutralizing antibodies with specificity for the host cell receptor/coreceptor complex (WO 97/46697), are not immunogens and cannot be used as preventative vaccines. They are agents for passive immunization only. A vaccine that acts by inducing an active anti-self antibody response against the receptor/coreceptor complex by active immunization, which would provide effective and long term protection from infection via the infrequent and convenient administration of small quantities of immunogen, would be far more preferable.
For efficacy, the immunogenic components of such an immunogenic composition must comprise a "B cell" epitope that mimics relevant sites on the host cell receptor/coreceptor complex with fidelity, or, a neighboring site if a mimetic peptide epitope cannot be identified, sufficient to evoke cross-inhibitory antibodies while retaining site-specificity sufficient to avoid adverse immunosuppression. The identification of such sites for mimicry by synthetic antigens has been difficult as the broadly neutralizing anti-receptor/coreceptor monoclonal antibodies reported previously recognize discontinuous conformational sites on CD4 that cannot be readily duplicated (WO 97/46697). Also, the reproduction of useful host cell antigenic target sites as portions of long recombinant immunogens cannot be readily applied as a means to avoid the need for exact knowledge of the vulnerable sites. For example, high titer hyperimmune antiserum to rsCD4 is devoid of neutralizing activity for primary isolates of HIV (WO 97/46697). The available 3D model for CD4 (as shown in http:www.pdb.bnl.gov/pdb.bin/pdbids) does not disclose useful immunogens for the host cell surface CD4/coreceptor complex. There remains a need for CD4/coreceptor immunogens, of relevant and safe site-specificity.
In addition to appropriate site-specificity, the receptor/coreceptor immunogens of an effective HIV vaccine must be highly immunostimulatory so as to evoke antibody responses of sufficient level for protection. These immunogens must also be designed to overcome the strong tolerance exhibited towards self molecules. Thus, there also remains a need for immunogens of sufficient immunopotency.
It is an object of the present invention to provide peptide compositions, having the desired site-specificity and immunopotency, as immunogens for the prevention of HIV infection.
Improved immunogenicity and appropriate specificity for the useful synthetic peptide immunogens of the present invention have been accomplished through incorporation of a collection of methods for the identification and design of synthetic peptide immunogens. These methods include: (1) an effective procedure for the identification of an effective high affinity target epitope; (2) the means for stabilization of the conformational features of that target site on a synthetic peptide by the introduction of cyclic constraints, so as to maximize cross-reactivity to the native molecule; (3) the means to augment the immunogenicity of the B cell target epitope by combining it with a site comprising a broadly reactive promiscuous T helper cell (Th) epitope; and (4) the means of enlarging the repertoire of T cell epitopes by application of combinatorial peptide chemistry and thereby further accommodate the variable immune responsiveness of an outbred population.
Synthetic peptides have been used for "epitope mapping" to identify immunodominant determinants or epitopes on the surface of proteins for the development of new vaccines and diagnostics. Epitope mapping employs a series of overlapping peptides corresponding to regions on the protein of interest to identify sites which participate in antibody-immunogenic determinant interaction. Commonly, epitope mapping employs peptides of relatively short length to precisely detect linear determinants. A fast method of epitope mapping known as PEPSCAN is based on the simultaneous synthesis of hundreds of overlapping peptides, of lengths of 8 to 14 amino acids, coupled to solid supports. The coupled peptides are tested for their ability to bind antibodies. The PEPSCAN approach is effective in localizing linear determinants, but not for the identification of epitopes needed for mimicry of discontinuous effector sites such as the HIV receptor/coreceptor binding site (Meloen et al., Ann Biol. Clin., 1991; 49:231-242). An alternative method relies on a set of nested and overlapping peptides of multiple lengths ranging from 15 to 60 residues. These longer peptides can be reliably but laboriously synthesized by a series of independent solid-phase peptide syntheses, rather than by the rapid and simultaneous PEPSCAN syntheses. The resulting set of nested and overlapping peptides can then be used for analyses of antibody binding in systems such as experimental immunizations and natural infections, to identify long peptides which best present immunodominant determinants, including simple discontinuous epitopes. This method is exemplified by the studies of Wang for the mapping of immunodominant sites from HTLV I/II (U.S. Pat. No. 5,476,765) and HCV (U.S. Pat. No. 5,106,726); and it was used for the selection of a precise position on the gp120 sequence for optimal presentation of an HIV neutralizing epitope (Wang et al., Science, 1991; 254:285-288).
Peptide immunogens are generally more flexible than proteins and tend not to retain any preferred structure. Therefore it is useful to stabilize a peptide immunogen by the introduction of cyclic constraints. A correctly cyclized peptide immunogen can mimic and preserve the conformation of a targeted epitope and thereby evoke antibodies with cross-reactivities for that site on the authentic molecule. For example, a loop structure present on an authentic epitope can be more accurately duplicated on a synthetic peptide by the addition of advantageously placed cysteine residues followed by cyclization through the sulfhydryl groups (Moore, Chapter 2 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman and Company: New York, 1992, pp. 63-67).
Another important factor affecting the immunogenicity of a peptide immunogen derived from a receptor/coreceptor complex is the presentation of this peptide to the immune system by T helper cell epitopes that react with a host's T-helper cell receptors and Class II MHC molecules (Babbitt et al., Nature, 1985; 317:359-361). T helper epitopes (Th) are often provided by carrier proteins with concomitant disadvantages due to the difficulties for the manufacture of well-defined peptide-carrier conjugates, misdirection of most antibody response to the carrier, and carrier-induced epitopic suppression (Cease, Intern Rev Immunol, 1990; 7:85-107; Schutze et al., J Immunol, 1985; 135:2319-2322). Alternatively, T cell help may be stimulated by synthetic peptides comprising Th sites. Thus, Class II Th epitopes termed promiscuous Th evoke efficient T cell help and can be combined with synthetic B cell epitopes that by themselves are poorly immunogenic to generate potent peptide immunogens (U.S. Pat. No. 5,759,551). Well-designed promiscuous Th/B cell epitope chimeric peptides are capable of eliciting Th responses and resultant antibody responses targeted to the B cell site in most members of a genetically diverse population expressing diverse MHC haplotypes. Promiscuous Th can be provided by specific sequences derived from potent foreign antigens, such as for example measeles virus F protein, hepatitis B virus surface antigen, and Chlamydia trachomatis major outer membrane protein (MOMP). Many known promiscuous Th have been shown to be effective in potentiating a poorly immunogenic peptide corresponding to the decapeptide hormone (U.S. Pat. No. 5,759,551).
Promiscuous Th epitopes range in size from about 15 to about 40 amino acid residues in length (U.S. Pat. No. 5,759,551), and often share common structural features and may contain specific landmark sequences. For example, a common feature is amphipathic helices, which are alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and with charged and polar resides dominating the surrounding faces (Cease et al., Proc Natl Acad Sci USA, 1987; 84:4249-4253). Th epitopes frequently contain additional primary amino acid patterns such as a Gly or charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue. This pattern defines what are called Rothbard sequences. Also, Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue, consistent with an amphipathic helix having positions 1, 4, 5 and 8 located on the same face. Since all of these structures are composed of common hydrophobic, charged and polar amino acids, each structure can exist simultaneously within a single Th epitope (Partidos et al., J Gen Virol, 1991; 72:1293-99). Most, if not all, of the promiscuous T cell epitopes contain at least one of the periodicities described above. These features may be incorporated into the designs of "idealized artificial Th sites".
Promiscuous Th epitopes derived from foreign pathogens include as examples, but are not limited to, hepatitis B surface and core antigen helper T cell epitopes (HB.sub.s Th and HB.sub.c Th), pertussis toxin helper T cell epitopes (PT Th), tetanus toxin helper T cell epitopes (TT Th), measles virus F protein helper T cell epitopes (MV.sub.F Th), Chlamydia trachomatis major outer membrane protein helper T cell epitopes (CT Th), diphtheria toxin helper T cell epitopes (DT Th), Plasmodium falciparum circumsporozoite helper T cell epitopes (PF Th), Schistosoma mansoni triose phosphate isomerase helper T cell epitopes (SM Th), and Escherichia coli TraT helper T cell epitopes (TraT Th). The pathogen-derived Th were listed as SEQ ID NOS:2-9 and 42-52 in U.S. Pat. No. 5,759,551; as Chlamydia helper site P11 in Stagg et al., Immunology, 1993; 79; 1-9; and as HBc peptide 50-69 in Ferrari et al., J Clin Invest, 1991; 88: 214-222.
Useful Th sites may also include combinatorial Th that incorporate selected degenerate sites into the design of the idealized Th sites. In Wang et al.(WO 95/11998), a particular class of a combinatorial epitope was designated as a "Structured Synthetic Antigen Library" or SSAL. A Th constructed as an SSAL epitope is composed of positional substitutions organized around a structural framework of invariant residues. The sequence of the SSAL is determined by aligning the primary amino acid sequence of a promiscuous Th, retaining relatively invariant residues at positions responsible for the unique structure of the Th peptide and providing degeneracy at the positions associated with recognition of the diverse MHC restriction elements. Lists of invariant and variable positions and preferred amino acids are available for MHC-binding motifs (Meister et al., Vaccine, 1995; 13:581-591).
All members of the SSAL are produced simultaneously in a single solid-phase peptide synthesis in tandem with the targeted B cell epitope and other sequences. The Th library sequence maintains the structural motifs of a promiscuous Th and accommodates reactivity to a wider range of haplotypes. For example, the degenerate Th epitope described as SSAL1TH1 was modeled after a promiscuous epitope taken from the F protein of measles virus (Partidos et al., 1991). SSAL1TH1 was used in tandem with an LHRH target peptide. Like the measles epitope, SSAL1TH1follows the Rothbard sequence and the 1, 4, 5, 8 rule:
1 5 10 15 Asp-Leu-Ser-Asp-Leu-Lys-Gly-Leu-Leu-Leu-His-Lys-Leu-Asp-Gly-Leu SEQ ID NOS:61 Glu Ile Glu Ile Arg Ile Ile Ile Arg Ile Glu Ile SEQ ID NOS:62 Val Val Val Val Val Val Val SEQ ID NOS:63 Phe Phe Phe Phe Phe Phe Phe SEQ ID NOS:64
Charged residues Glu or Asp are added at position 1 to increase the charge surrounding the hydrophobic face of the Th. The hydrophobic face of the amphipathic helix is then maintained by hydrophobic residues at 2, 5, 8, 9, 10, 13 and 16, with variability at 2, 5, 8, 9, 10, 13 and 16 to provide a facade with the capability of binding to a wide range of MHC restriction elements. The net effect of the SSAL feature is to enlarge the range of immune responsiveness to an artificial Th (WO 95/11998).
Peptide immunogens that have been designed with the peptide technologies and peptide design elements discussed above, i.e., precise epitope mapping, cyclic constraint, and the incorporation of promiscuous Th epitopes or idealized promiscuous Th, and idealized SSAL Th epitopes, are the basis for the effective synthetic receptor/coreceptor complex vaccines for HIV of the present invention. Such peptides are preferred for appropriate targeting and safety due to effective presentation of a portion of the HIV receptor/coreceptor binding site by optimized positioning and cyclization, and for immunopotency due to broadly reactive Th responsiveness.